Bacterial DNA replication is carefully controlled at the initiation stage, largely by regulation of-the essential activity of DnaA protein. The cellular membrane has long been hypothesized to be involved in chromosomal replication, with accumulating evidence indicating membranes have a profound influence on DnaA protein. DnaA protein is found at the cell membrane in vivo, and membranes containingacidic phospholipids can convert an inert form of DnaA protein into replicatively active DnaA. To prevent re-initiation within the same cell-cycle, the membrane-associated protein Hda converts active DnaA into its inactive form commensurate with DNA replication. Cells depleted of acidic phospholipids undergo growth-arrest, which certain mutant forms of DnaA protein are able to suppress, further implicating a close link between initiation of replication and membrane acidic phospholipids. Formation of acidic phospholipid domains at specific sites on the cell membrane occurs in a cell-cycle dependent manner, related to the fixed sites where DNA replication occurs. The long-term goal of this research is to elucidate the physiological significance of the influence membranes have on chromosomal replication. The research outlined here uses biochemical approaches with defined components, physiological studies, cytolocalization techniques, and structural studies to answer whether membrane domains of acidic phospholipids temporally and spatially control the initiationactivity of DnaA protein. The Specific Aims are to: 1. Test the hypothesis that an inadequate level of acidic phospholipids arrests the cell-cycle at the initiation of chromo-somal replication. Examine the cell-cycle parameters of synchronized cells to determine whether cells arrest specifically at the time of initiation as they are depleted of acidic phospholipids. Use in vivo footprintingof oriC to determine whether the absence of acidic phospholipids prevents DnaA from properly forming the pre-replication complexes that are necessary for initiation to occur. 2. Test the hypothesis that membrane domains enriched in acidic phospholipids activate membrane-bound DnaA for initiation at the correct location within the cell and period of the cell-cycle. Use fluorescence microscopy of living cells to examine the importance of acidic phospholipids for the membrane localization of DnaA, and whether the cell-cycle related appearance of an acidic phospholipid domain at the future site of chromosomal replication activates DnaA to initiate the formation of a replisome. Employ FRET analysis to determine whether acidic phospholipids are needed for DnaA-Hda interactionat the replisome. 3. Define the structure of DnaA protein bound to acidic lipid bilayers. Use the lipidic cubic phase in meso method to obtain diffraction-quality crystals for high-resolution structural determination of DnaA protein associated with a lipid bilayer that contains acidic phospholipids. This work should provide insight into the interactions between membrane lipids and DNA synthesis components, and the functions that membranes may play in cell-cycle and growth controlled initiation of chromosomal replication. Furthermore, advances in understanding of how membrane lipids influence DnaA may help direct future investigations in the emerging field of phospholipid-mediated regulation of enzymaticactivities.